1. Field of the Invention
The field of the invention relates to methods of producing a recombinant fibroblast growth factor protein and its use in promoting angiogenesis.
2. Description of the Related Art
Fibroblast growth factors (FGF) are nine structurally related polypeptides, which are potent regulators of cell proliferation, differentiation and normal development. They also take part in pathological processes of tumorogenesis and metastasis (Galzie, et al. Biochem. Cell Biol. (1997) 75:669–685). They are potent mitogens and differentiation factors for a broad range of mesoderm and neuroectoderm derived cells, including endothelial cells.
The heparin proteoglycans, heparin or heparin sulfate, bind several FGF molecules together as a complex which are presented to the FGF receptors. FGF proteins bind to their receptors resulting in the activation of protein tyrosine kinases. The phosphorylation of these tyrosine kinases initiates multiple signals including the transcription of new mRNA's.
Two fibroblast growth factors, basic and acidic, are described as potent inducers of angiogenesis (Friesel et al. (1995) FASEB J. 9:919–925). Both basic and acidic factors have been implicated in the control of blood vessel formation and their involvement in normal and pathological angiogenesis (Slavin, J. (1995) Cell Biology International 19(5): 431–444). These factors have been purified, their amino acid sequences have been determined and their cDNA has been cloned and sequenced.
Acidic Fibroblast Growth Factor (aFGF) has been described under various names including embryonic kidney-derived angiogenesis factor I, astroglial growth factor I, endothelial cell growth factor (ECGF), retina-derived growth factor, heparin-binding growth factor class 1, endothelial growth factor, eye-derived growth factor II, prostatropin, and glial maturation factor (Gospodarowicz, et al. (1987) Journal of Cellular Physiology supplement 5: 15–26). Cloning, nucleotide sequence and chromosome localization have been described (Jaye et al. (1986) Science 233:541–545).
Acidic fibroblast growth factor (aFGF or FGF-1) belongs to a large family of heparin-binding growth factors that are now generally referred to as fibroblast growth factors (FGFs). To date there are at least 22 known members of the FGF family, with FGF-1 and FGF-2 (basic FGF, bFGF) as its prototype members. Besides vascular endothelial growth factor (VEGF), FGF-1 is well known as a highly potent angiogenic agent with mitogenic activity for a wide variety of different cell types as well in tissue culture as in vivo. The biological process of angiogenesis is initiated by binding of FGF-1 to the specific receptor molecules (FGFR) located on the cell surface. FGFR activation is followed by tyrosine autophosphorylation—thus initiating the complex cascade of matrix dissolution, cell differentiation and proliferation, finally resulting in tube formation and new vessel growth.
The aFGF gene is situated on chromosome 5. It has a single copy and encodes three exons separated by two introns. A 4.8 kb mRNA translates synthesis of a form of aFGF with 155 amino acids. However, the N-terminal methionine residue is removed in vivo to give a 154 amino acid form. This 154 amino acid form of the aFGF is processed into two forms which are 140 and 134 amino acids. The aFGF protein is an anionic mitogen of molecular weight 15,000–17,000 D.
The aFGF protein has been found in brain, retina, bone matrix and osteosarcoma. Only forms with 140 and 134 amino acids have been obtained from tissues. It has been suggested that the truncated aFGF forms are an artifact created by specific proteases during aFGF extraction and isolation (Gospodarowicz, et al. (1987) Journal of Cellular Physiology supplement 5:15–26; Jaye et al. (1987) The Journal of Biological Chemistry 262 (34):16612–16617).
It has been suggested that heparin potentiates the biological activity of the aFGF protein (Thornton et al. (1983) Science 222 (4624): 623–625). Heparin binding to aFGF has been observed (Maciag et al. (1984) Science 225 (4665): 932–935). This heparin-binding characteristic has been used as an efficient affinity chromatography method for the purification of aFGF protein. Heparin potentiates the biological activity of aFGF and the enhanced activity of the aFGF-heparin complex varies from several to one hundred fold (Lobb, et al. (1986) Anal. Biochem. 154:1–14).
In one embodiment, the present invention is related to the treatment of coronary heart disease by revascularization therapy, and more particularly to pharmaceutical compositions containing recombinant fibroblast growth factor, procedures for preparing recombinant fibroblast growth factor, and methods for delivering the pharmaceutical compositions containing a fibroblast growth factor to the ischemic myocardium.
Heart attack, or myocardial infarction, due to coronary heart disease (CHD) is the single leading cause of death in the U.S. according to the American Heart Association. Myocardial infarction occurs when the blood supply to part of the heart muscle, or myocardium, is severely reduced or stopped, thereby depriving the myocardium of oxygen. This oxygen deprivation, or ischemia, occurs when one of the coronary arteries which supply blood to the myocardium is blocked. The blockage, or stenosis, most frequently results from atherosclerosis, a condition associated with the buildup of fatty deposits in the vessel walls. Statistics based upon the National Heart, Lung, and Blood Institute's Atherosclerotic Risk in Communities (ARIC) Study (1987–1994) and the Framingham Heart Study, indicate that the CHD-related mortality rate in the U.S. is one of every 4.8 deaths (481,287 deaths in 1995). Over one million new and recurrent cases of heart attack and almost 14 million victims of myocardial ischemia, angina and other manifestations of CHD (7.1 million men and 6.8 million women) are reported each year. Moreover, as many as 3 to 4 million individuals in the U.S. alone may have ischemic episodes (silent ischemia) without knowing it.
Procedures currently available for treating CHD and myocardial ischemia include: 1) coronary artery bypass graft, wherein a segment of a vein is harvested from the patient's leg and grafted in such a manner as to reroute blood around the stenosis; 2) percutaneous transluminal coronary angioplasty, or balloon angioplasty, wherein a catheter having a deflated balloon is passed into the stenosed region of the artery and the balloon is then inflated to widen the vessel lumen; 3) laser angioplasty, wherein a catheter having a laser at its distal tip is used to ablate the atherosclerotic plaque; 4) artherectomy, wherein a high-speed rotating ‘burr’ at the end of a catheter is used to grind away the atherosclerotic plaque; and 5) transmyocardial revascularization, in which a series of channels are cut in the myocardium by laser to allow blood from inside the left ventricle to permeate into the ischemic heart muscle. While variations, combinations and improvements in these basic approaches are constantly being developed, each of these alternative methods have significant disadvantages.
Thoracic surgeons performed approximately 573,000 bypass operations in 1995 in the U.S. alone. While coronary artery bypass has the advantage of creating a new path through which blood may flow freely to the myocardium, often by graft directly from the aorta or internal mammary artery, it also has the major disadvantage of requiring highly invasive open heart surgery. Indeed, the heart is generally stopped in bypass surgery to facilitate anastomosis of the graft to the coronary artery. Oxygenation and circulation are maintained by a heart-lung machine. Consequently, bypass patients face increased risk of damage to the kidneys, brain and other organs. In addition to the medical risks, bypass procedures are very expensive and require significant recovery time. Moreover, for many patients who are at high risk for major invasive surgery or who have advanced stage and/or diffuse CHD, coronary artery bypass procedures are not a viable option. Consequently, these patients must seek alternative treatments.
The most frequently utilized, less invasive alternative to bypass surgery, is percutaneous transluminal coronary angioplasty, commonly referred to as balloon angioplasty. Approximately 434,000 balloon angioplasties were performed in the U.S. in 1995. While such procedures are considerably less invasive and less expensive than coronary bypass surgery, the improvement in blood flow to the myocardium may be small and short-lived For instance, according to the American Heart Association, an increase in luminal diameter of greater than 20% is considered successful. Furthermore, restenosis occurs within six months in about 25–30% of patients who have undergone successful angioplasty. To reduce the incidence of restenosis following angioplasty, expandable structural supports, referred to as stents, may be deployed during angioplasty to maintain vessel diameter and blood flow. However, the endothelial and smooth muscle cells which comprise the vessel walls tend to infiltrate the stent scaffolding, eventually compromising blood flow. Finally, balloon angioplasty is not recommended for patients with severe diffuse CHD or in patients having greater than 50% occlusion in their left anterior descending (LAD) coronary artery. Thus, balloon angioplasty is neither sufficiently effective nor widely applicable to alleviate the debilitating symptoms of severe myocardial ischemia in many patients.
Two other catheter-based techniques, laser angioplasty and arthrectomy, are directed toward increased blood flow through removal of atherosclerotic plaque. While these techniques may be used alone, they are often used in conjunction with balloon angioplasty to increase luminal diameter. Unfortunately, plaque removed by these methods may generate debris and/or flaps which may cause sudden, dangerous post-operative occlusions.
Transmyocardial revascularization, or laser revascularization, is another procedure, which is both less invasive and less costly than bypass surgery, and has been forwarded as an option for those patients who are at high risk for a second bypass or angioplasty. By providing direct access of the ischemic myocardium to blood within the ventricular chamber, laser revascularization may be useful in treating patients whose coronary artery blockages are too diffuse to be treated effectively with site-directed bypass surgery and/or angioplasty. Unfortunately, the theoretical benefits of laser revascularization have yet to be proven safe and effective over time. Indeed, the generation of an array of channels cut through the walls of the heart by laser vaporization may serve merely as a stop-gap measure to address acute myocardial ischemia, while diminishing the long-term prognosis.
Thus, there remains a substantial gap in treatment options for CHD patients, particularly those who are at high risk for bypass surgery. Indeed, there is a need for a treatment protocol which is less invasive and less expensive than bypass surgery, and more effective than balloon angioplasty and transmyocardial revascularization.
Normal capillaries have a cell population with a low turnover rate of months or years. On occasion, however, a high turnover rate of this cell population is possible even under physiological conditions, and this naturally leads to the rapid growth of new capillaries and other blood vessels. Such a physiological process occurs in the development of the placenta, in fetal growth, and in wound healing, as well in the formation of collaterals in response to tissue ischemia. Angiogenic polypeptide growth factors are essential for such processes as capillary growth or neoangiogenesis. These growth factors bring about their effects by significantly increasing cell proliferation, differentiation, and migration via high-affinity receptors on the surfaces of the endothelial cells. Accordingly, the present invention is directed toward revascularization of the myocardium via local-acting, growth factor-stimulated neoangiogenesis.